Mammalian spermatogenesis is a highly ordered process of cell differentiation producing haploid spermatozoa with a number of unique structures and molecular constituents. The regulation of this process is not well defined, although it is clear that differentiation proceeds with precise kinetics and involves complex interactions between germ cells, Sertoli cells and other somatic cells in the testis. The objective of this research proposal is to determine roles of the cation-independent (Cl) and cation-dependent (CD) mannose 6-phosphate (M6P) receptors in the regulation of spermatogenesis. In addition to the well-defined role of M6P receptors in targeting acid hydrolases to lysosomes, the Cl M6P receptor has distinct binding sites for M6P and for insulin-like growth factor II (IGF-II) and may mediate growth factor effects. Mouse pachytene spermatocytes and round spermatides synthesize predominantly the CD receptor and lower levels of the Cl receptor. Sertoli cells in culture, unlike germ cells or any other cell type to our knowledge, synthesize the Cl M6P receptor almost exclusively. This differential expression suggests that the two M6P receptors mediate interactions between germ cells and Sertoli cells and/or participate in targeting hydrolytic enzymes to the acrosome. In Specific Aim 1, monospecific antibodies and DNA probes will be used to localize the Cl and CD receptors in germ cells and determine if their expression undergoes stage-specific variations during the cycle of the seminiferous epithelium. Isolated cells will be used to monitor the transcription and translation of M6P receptors at other spermatogenic stages and to determine if these receptors are localized in distinct subcellular locations during germ cell differentiation and in mature sperm. Specific Aim 2 will test the hypothesis that M6P receptors participate in germ cell-Sertoli cell interactions mediated by secretory products (M6P-containing glycoproteins or IGF-II). Preliminary studies have shown that germ cells and Sertoli cells have surface M6P receptors that mediate endocytosis, and that Sertoli cells secrete M6P-containing glycoproteins that are endocytosed by germ cells. Effects of Sertoli M6P-glycoproteins or IGF-II on germ cell function via signal transduction mechanisms or endocytosis will be assessed. Immunoblot analyses will be used to determine if the M6P- glycoprotein fraction contains known Sertoli proteins and growth factors. In addition, it will be determined if isolated germ cells secrete M6P- containing glycoproteins or IGF-II. To assess the potential role of M6P receptors in targeting acid hydrolases to the acrosome, oligosaccharide structural analyses will be conducted to determine if newly synthesized acrosomal constituents acquire the M6P recognition marker during post- translational processing (Specific Aim 3). In addition, transgenic mice will be used to determine if N-glycosylation is required for acrosomal targeting. By examining the roles of M6P receptors in the local regulation of spermatogenesis, these studies may provide new insights for the development of contraceptives, the clinical management of infertility, and the assessment of reproductive toxicants in the male.